Figure 3.

K10 as a cold-inducible target of Raynaud’s-associated antibodies. Cultured cells were kept at 37°C in complete medium (see Methods). Where indicated for cold exposure, cells were exposed to 4°C medium for 1 minute 24 hours before cell harvesting, then returned to 37°C. A. Anti-endothelial activity by immunofluorescence of HUVEC (see Methods) with an RNP+ serum from a Raynaud’s patient. B. Reverse immunophenotyping immunoprecipitation (see Methods) of HUVEC lysates with an anti-RNP+ human Raynaud’s Phenomenon serum identified a ~56kD band (arrow) that was not pre-cleared by an RNP+ serum from a patient without Raynaud’s Phenomenon (RP-), for MSMS analysis. C. Expression and cold-induced upregulation of mRNA levels by real time PCR were quantitated for K10 and control cytokeratin 18 (K18), normalized to expression of 18S ribosomal RNA. The endothelial cell line HUVEC expressed much higher levels of K10 message than epithelial HEK293 cells or lymphoid Jurkat cells at 37°C, and further dramatically upregulated K10 but not K18 expression levels after cold exposure. D. Expression and upregulation of K10 protein in HUVEC. While low levels of K10 protein were present in HUVEC cells at 37°C, the quantity of protein dramatically increased in cold-exposed cells (compared to GAPDH loading control).